Objectives of field studies in Mali 1. Characterize the epidemiology of malaria in Kenieroba, Mali 2. Investigate the impact of hemoglobin (HbAS and HbAC) and other host red cell polymorphisms on malaria incidence and disease 3. Characterize malaria-specific antibody responses in Kenieroba children and adults 4. Explore mechanisms involved in malaria pathogenesis 5. Investigate opsonization as a host effector mechanism against malaria infected red cells focusing on antibodies to VAR2CSA in multigravid women In collaboration with Dr. Rick Fairhurst (LMVR) and Dr. Mahamadou Diakite(Mali ICER), we have developed an epidemiologic profile of 1500 children in Kenieroba, Mali, over a 4-year period and we are accumulating this information for publication. This is probably the most detailed longitudinal study of malaria in Africa that has been conducted. Also all the children have been genotyped for a series of red cell and hemoglobin polymorphisms (including sickle cell trait HbAS) that may influence susceptibility to malaria. In 2009 we identified a sub-cohort of these children, selecting those with the sickle cell trait (HbAS) and pairing them with age-matched HbAA controls and we have followed these children since that time. Blood samples being obtained before and after the transmission season as well as during bouts of clinical malaria infection; these samples provide a unique and valuable resource for future studies on the development of humoral and cellular responses to blood-stage antigens of malaria parasites. As anticipated, our data show a progressive increase in resistance to malaria incidence with age. Interestingly, children with HbAS but not HbAC genotypes have shown significant protection against uncomplicated malaria. Because antibodies have been implicated as important contributors to the progressive acquisition of immunity, we continue to conduct detailed analysis of the longitudinal changes in anti-malarial antibodies in these children. We have shown that the resistance of HbAS children cannot be attributed to higher levels of antibodies to merozoite antigens. Additionally, because there has been long-standing data suggesting that naturally acquired immunity arises in part from repeated exposure to strains of parasites expressing different variant antigens on the surface of infected erythrocytes (IEs), we have developed a flow cytometry assay to measure such antibodies. Again, we could not show that these antibodies were the basis of the resistance of HbAS children and we are seeking other, more physiologic explanations. Pregnancy associated malaria (PAM) remains a major threat to women and their unborn children in endemic areas. IEs accumulate in the placenta and the ensuing inflammatory response can increase the likelihood of anemia, hypertension, premature delivery and possible death of low birth weight infants. Clinical immunity to PAM in multigravid women has been attributed to antibodies that recognize VAR2CSA, a large multi-domain variant of the PfEMP-1 family which binds to chondroitin sulfate A (CSA), on the surface of red cells. However, there is no agreement as to the relative importance of its 6 Duffy binding-like domains. Opsonization of infected erythrocytes by cytophilic antibodies that recognize VAR2CSA epitopes represents an understudied host effector mechanism in PAM. We have established a flow cytometry-based opsonization assay and we have shown that, in contrast to malaria-nave American adults and malaria-exposed Malian men, purified IgGs from multigravid Malian women showed higher 1) reactivity to recombinant DBL domains by ELISA, 2) binding to red cells expressing VAR2CSA, and 3) opsonization of these infected erythrocytes by human monocytic cells. Importantly, preincubation of IgGs from multigravid women with selected VAR2CSA domains significantly diminished opsonization of VAR2CSA-expressing IEs by monocytes. Antigen reversal of opsonization provides the first evidence that domains DBL3x, DBL5&#949;, and DBL2x, are the primary targets of these opsonizing antibodies. Our study focuses attention on these domains for PAM vaccine development and introduces a new tool to identify targets of host effector responses to pathogens. Finally we have collaborated on studies on parasite clearance time in Malian children treated with artemisinin and on studies of malaria pathogenesis (uric acid and microparticles) in the population of Malian children. Objectives of studies on potential P. falciparum targets of immunity 1. Search for conserved, parasite-encoded epitopes on the surface of malaria IEs and in merozoites 2. Test new approaches to problems with existing blood-stage vaccine candidates 3. Assess the potential of RBL and EBL families as vaccine candidates We have taken a novel approach using DNA aptamers to look for conserved, parasite-encoded epitopes on the surface of IEs as well as using immunoproteomics to look for novel merozoite antigens which might elicit in vitro parasite growth inhibition activity (GIA). We have identified DNA sequences which bind to IEs with nanomolar dissociation constants, and a subset of aptamers recognized all laboratory-adapted clones and field isolates of P. falciparum tested. We are using the aptamers to identify their targets from parasitized red cells extracts. We have also addressed the antigenic polymorphism associated with a traditional vaccine candidate, PfAMA1 (apical membrane antigen-1), showing that a mixture of 5 different AMA1 alleles overcomes allelic polymorphism. In addition to a better understanding of traditional blood-stage antigens such as PfAMA1 and PfMSP1 (merozoite surface protein-1), recent results with two different merozoite protein families - the reticulocyte binding-like family (RBL) and the erythrocyte binding-like (EBL) proteins have given new impetus to this area. Results with P. falciparum RH5 are particularly encouraging in eliciting antibodies with strong GIA activity and support pursuing this as a vaccine candidate. Objectives of work on parasite sexual stages and malaria transmission 1. Develop quantitative methodology for analysis of the SMFA to evaluate transmission blocking activity 2. Determine whether Kenieroba residents develop antibodies which have blocking activity in SMFA 3. Assess the presence of sexual stage parasites in Kenieroba residents During the past few years increased attention has been focused on the sexual stages of the parasite and their development in the mosquito vector as part of long-range goals of malaria elimination and eradication. The gold standard assay to evaluate the ability of antibodies to block transmission to mosquitoes is the standard mosquito membrane feeding assay (SMFA). We have performed an in depth study of this assay to define its characteristics in order to have confidently assess potential transmission blocking vaccine (TBV) candidates. We have shown that it is quite reproducible at high concentrations of antibody but highly variable at low concentrations. We have worked with the Biostatistics group at NIAID to develop a computer model of the assay. Only a few proteins have been tested as potential TBV and we are searching for new targets and evaluating antibodies to the targets identified by others. In the spring of 2013 we initiated a new study (NIH 13-I-N107) of malaria transmission in a field study in Kenieroba to address the limited information on actual transmission in malaria endemic areas. We are evaluating the carriage of gametocytes in 500 villagers of all ages in this population. Volunteers are being finger pricked twice per month for collection of DNA and RNA. This will allow us to determine whether they have blood stage parasites and/or sexual stage parasites. We will also genotype the parasite populations as they adapt to the long dry season.